Christophe Hermans

Ultrasound-guided approach to nerves (direct vs. tangential) and the incidence of intraneural injection: a cadaveric study

This study evaluated the incidence of nerve puncture and intraneural injection based on the needle approach to the nerve (direct vs. tangential). Two expert operators in regional anaesthesia performed in‐plane ultrasound‐guided nerve blocks (n = 158) at different levels of the brachial plexus in cadavers, aiming either directly for the nerve (n = 77) or tangentially inferior to the nerve (n = 81). After reaching the outer limit of the nerve, the needle was intentionally advanced approximately 1 mm in both approaches, and 0.2–0.5 ml of saline was injected. Each operator classified (in real time) the needle tip and injectate as intraneural or not. Video clips showing the final position of the needle and the injection were evaluated in the same manner by seven independent expert observers who were blinded to the aims of this study. In addition, 20 injections were performed with ink for histological evaluation. Intraneural injections of saline were observed by the operator in 58% (45/77) of cases using the direct approach and 12% (10/81) of cases using the tangential approach (p < 0.001). The independent observers agreed with the operator in a substantial number of cases (Cohens kappa index 0.65). Histological studies showed intraneural spread in 83% (5/6) of cases using the direct approach and in 14% (2/14) of cases using the tangential approach (p = 0.007). No intrafascicular injections were observed. There was good agreement between the operators’ assessment and subsequent histological evaluation (Cohens kappa = 0.89). Simulation of an unintentional/accidental advancement of the needle ‘beyond the edge’ of the nerve suggests significantly increased risk of epineural perforation and intraneural injection when a direct approach to the nerve is used, compared with a tangential approach.

Deep sequencing of the TP53 gene reveals a potential risk allele for non–small cell lung cancer and supports the negative prognostic value of TP53 variants

The TP53 gene remains the most frequently altered gene in human cancer, of which variants are associated with cancer risk, therapy resistance, and poor prognosis in several tumor types. To determine the true prognostic value of TP53 variants in non–small cell lung cancer, this study conducted further research, particularly focusing on subtype and tumor stage. Therefore, we determined the TP53 status of 97 non–small cell lung cancer adenocarcinoma patients using next generation deep sequencing technology and defined the prognostic value of frequently occurring single nucleotide polymorphisms and mutations in the TP53 gene. Inactivating TP53 mutations acted as a predictor for both worse overall and progression-free survival in stage II–IV patients and patients treated with DNA-damaging (neo)adjuvant therapy. In stage I tumors, the Pro-allele of the TP53 R72P polymorphism acted as a predictor for worse overall survival. In addition, we detected the rare R213R (rs1800372, minor allele frequency: 0.0054) polymorphism in 7.2% of the patients and are the first to show the significant association with TP53 mutations in non–small cell lung cancer adenocarcinoma patients (p = 0.003). In conclusion, Our findings show an important role for TP53 variants as negative predictors for the outcome of non–small cell lung cancer adenocarcinoma patients, especially for TP53 inactivating mutations in advanced stage tumors treated with DNA-damaging agents, and provide the first evidence of the R213R G-allele as possible risk factor for non–small cell lung cancer.

Towards Prognostic Profiling of Non-Small Cell Lung Cancer: New Perspectives on the Relevance of Polo-Like Kinase 1 Expression, the TP53 Mutation Status and Hypoxia

Background: Currently, prognosis of non-small cell lung cancer (NSCLC) patients is based on clinicopathological factors, including TNM stage. However, there are considerable differences in patient outcome within a similar staging group, even when patients received identical treatments. In order to improve prognostic predictions and to guide treatment options, additional parameters influencing outcome are required. Polo-like kinase 1 (Plk1), a master regulator of mitotic cell division and the DNA damage response, is considered as a new potential biomarker in this research area. While several studies reported Plk1 overexpression in a broad range of human malignancies, inconsistent results were published regarding the clinical significance hereof. A prognostic panel, consisting of Plk1 and additional biomarkers that are related to the Plk1 pathway, might further improve prediction of patient prognosis. Methods: In this study, we evaluated for the first time the prognostic value of Plk1 mRNA and protein expression in combination with the TP53 mutation status (next generation sequencing), induction of apoptotic cell death (immunohistochemistry for cleaved caspase 3) and hypoxia (immunohistochemistry for carbonic anhydrase IX (CA IX)) in 98 NSCLC adenocarcinoma patients. Results: Both Plk1 mRNA and protein expression and CA IX protein levels were upregulated in the majority of tumor samples. Plk1 mRNA and protein expression levels were higher in TP53 mutant samples, suggesting that Plk1 overexpression is, at least partially, the result of loss of functional p53 (<0.05). Interestingly, the outcome of patients with both Plk1 mRNA and CA IX protein overexpression, who also harbored a TP53 mutation, was much worse than that of patients with aberrant expression of only one of the three markers (p=0.001). Conclusion: The combined evaluation of Plk1 mRNA expression, CA IX protein expression and TP53 mutations shows promise as a prognostic panel in NSCLC patients. Moreover, these results pave the way for new combination strategies with Plk1 inhibitors.

Conditional mouse models support the role of SLC39A14 (ZIP14) in Hyperostosis Cranialis Interna and in bone homeostasis

Hyperostosis Cranialis Interna (HCI) is a rare bone disorder characterized by progressive intracranial bone overgrowth at the skull. Here we identified by whole-exome sequencing a dominant mutation (L441R) in SLC39A14 (ZIP14). We show that L441R ZIP14 is no longer trafficked towards the plasma membrane and excessively accumulates intracellular zinc, resulting in hyper-activation of cAMP-CREB and NFAT signaling. Conditional knock-in mice overexpressing L438R Zip14 in osteoblasts have a severe skeletal phenotype marked by a drastic increase in cortical thickness due to an enhanced endosteal bone formation, resembling the underlying pathology in HCI patients. Remarkably, L438R Zip14 also generates an osteoporotic trabecular bone phenotype. The effects of osteoblastic overexpression of L438R Zip14 therefore mimic the disparate actions of estrogen on cortical and trabecular bone through osteoblasts. Collectively, we reveal ZIP14 as a novel regulator of bone homeostasis, and that manipulating ZIP14 might be a therapeutic strategy for bone diseases.

Mutation and methylation analysis of circulating tumor DNA can be used for the follow-up of metastatic colorectal cancer patients

Background Targeted therapies, although contributing to survival improvement in metastatic colorectal cancer (mCRC), are expensive and may cause adverse effects. Therefore, confirming that patients are responding to these therapies is extremely important. Currently, follow‐up is performed using radiographic evaluation, which has its limitations. Liquid biopsies, reflecting real‐time tumor characteristics, hold great potential in monitoring tumor disease. Patients and Methods Blood samples were collected at different time points during treatment of 24 mCRC patients. Mutation and NPY methylation picoliter droplet‐based digital PCR (ddPCR) assays were performed on circulating DNA to investigate whether these assays can be used for disease monitoring. Results The results of the mutation and methylation assays were correlated with each other and corresponded with the results of radiographic evaluation. There was a steep decrease in circulating tumor DNA levels immediately after treatment initiation. Furthermore, circulating tumor DNA levels were increased in progressive samples and were undetectable in patients undergoing curative surgery. Conclusion This prospective study showed that tumor‐specific mutation and NPY methylation ddPCR assays performed on circulating DNA can be used for the follow‐up of mCRC patients during treatment and could complement current follow‐up methods. The analysis of NPY methylation is promising, as it has the additional advantage that no prior knowledge of tumor mutations is needed. Micro‐Abstract Improvements in follow‐up methods of metastatic colorectal cancer patients during treatment are warranted to ensure response to therapy. The potential of circulating tumor DNA (ctDNA) for follow‐up was investigated using methylation and mutation analyses in different blood samples collected during treatment. Our results corresponded to the results of radiographic evaluation, and therefore the analysis of ctDNA is valuable to complement current follow‐up methods.

Preclinical data on the combination of cisplatin and anti-CD70 therapy in non-small cell lung cancer as an excellent match in the era of combination therapy

In contrast to the negligible expression of the immunomodulating protein CD70 in normal tissue, we have demonstrated constitutive overexpression of CD70 on tumor cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. This can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Early clinical trials of these antibodies have already shown promising results in CD70-positive malignancies.In this study, we explored the potential of cisplatin to induce CD70 expression in NSCLC. Using real-time measurement tools, we also assessed the efficacy of a combination regimen with cisplatin and anti-CD70 therapy under normoxia and hypoxia. We identified an induction of CD70 expression on lung cancer cells upon low doses of cisplatin, independent of oxygen levels. More importantly, the use of cisplatin resulted in an enhanced ADCC-effect of anti-CD70 therapy. As such, this combination regimen led to a significant decrease in lung cancer cell survival cell survival, broadening the applicability the applicability of CD70-targeting therapy.This is the first study that proves the potential of a combination therapy with cisplatin and CD70-targeting drugs in NSCLC. Based on our data, we postulate that this combination strategy is an interesting approach to increase tumor-specific cytotoxicity and reduce drug-related side effects.In contrast to the negligible expression of the immunomodulating protein CD70 in normal tissue, we have demonstrated constitutive overexpression of CD70 on tumor cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. This can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Early clinical trials of these antibodies have already shown promising results in CD70-positive malignancies. In this study, we explored the potential of cisplatin to induce CD70 expression in NSCLC. Using real-time measurement tools, we also assessed the efficacy of a combination regimen with cisplatin and anti-CD70 therapy under normoxia and hypoxia. We identified an induction of CD70 expression on lung cancer cells upon low doses of cisplatin, independent of oxygen levels. More importantly, the use of cisplatin resulted in an enhanced ADCC-effect of anti-CD70 therapy. As such, this combination regimen led to a significant decrease in lung cancer cell survival cell survival, broadening the applicability the applicability of CD70-targeting therapy. This is the first study that proves the potential of a combination therapy with cisplatin and CD70-targeting drugs in NSCLC. Based on our data, we postulate that this combination strategy is an interesting approach to increase tumor-specific cytotoxicity and reduce drug-related side effects.

Abstract 4328: New perspectives on the use of polo-like kinase 1 as a prognostic biomarker in non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Polo-like kinase 1 (Plk1) overexpression is observed in various tumors, including non-small cell lung cancer (NSCLC), and is correlated with poor patient prognosis and metastasis, suggesting a role in aggressive tumors. Previous studies reported Plk1 downregulation by P53 upon DNA damage, suggesting that TP53 mutations might have an influence on Plk1 expression. In this study, we determined the Plk1 protein level in NSCLC patients and correlated these results with the TP53 status, presence of hypoxia (carbonic anhydrase 9, CA-9) and apoptosis induction (cleaved caspase 3). Material & methods: Tumor tissues of 84 NSCLC patients and 16 control samples were obtained from the Antwerp University Hospital. Immunohistochemistry was performed using antibodies to Plk1 (208G4, 1/50), CA-9 (EPR4151(2), 1/350) and cleaved caspase 3 (9579S, 1/250). Based on the% positive cells and staining intensity, an overall score of negative, weak, moderate or strong expression was assigned for both the Plk1 and CA-9 protein. For the TP53 mutation analysis, DNA was isolated using the QIAamp® DNA FFPE tissue kit. For each sample, DNA concentration and purity were assessed using NanoDrop measurement. The relation between DNA input and FFPE derived DNA quality was determined using the QC-plex assay from Multiplicom. Samples were considered to be usable when the DNA quality coefficient was higher than 0.2. Determination of the TP53 mutation status was performed using Multiplicoms TP53 MASTRTM Plus test with MID for Illumina Miseq®. Results & discussion: Plk1 and CA-9 positivity was detected in 95% and 83% of all tumor samples, respectively. Of the 16 control samples, all samples stained negative, except for 4/16 and 1/16 samples that showed a low expression for Plk1 and CA-9, respectively. In a next phase, cleaved caspase 3 staining will be scored and both CA-9 and cleaved caspase 3 expression patterns will be correlated to Plk1 expression. Furthermore, results will be correlated with clinicopathological parameters, including incidence age, smoking behavior, tumor differentiation and stage, metastasis and survival. For the TP53 mutation analysis, sufficient DNA with acceptable purity was isolated from 82 patients. A QC plex reaction has already been performed for 20 samples, 12 of them showed an acceptable DQC value and 8 sampled were marked as low DNA quality samples. Nonetheless, the latter could be used for further analysis by increasing the DNA input. At present, 15 samples have been sequenced successfully. Besides polymorphisms, 5 exon mutations (c.536A>G, c.916C>T, c.734G>A, c.578A>C, c.559+1G>T) and 4 intron mutations (c.920-2A>T, c.993+352C>T, c.559+1G>T) were observed. As soon as all samples have been processed, it will be evaluated whether a link between Plk1 overexpression and TP53 mutations can be evidenced. Citation Format: Jolien Van den Bossche, Filip Lardon, Christophe Hermans, Christophe Deben, Vanessa Deschoolmeester, Julie Jacobs, Karlijn van der Ven, Jurgen Del-Favero, Patrick Pauwels, Marc Peeters, An Wouters. New perspectives on the use of polo-like kinase 1 as a prognostic biomarker in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2015-4328

Abstract 3563: Unlocking the potential of CD70 as a therapeutic target in non-small cell lung cancer

Introduction: Upon interaction of CD70, a type II transmembrane protein, with its receptor CD27, the NFκB pathway is activated, leading to proliferation and survival. Hence, CD70 expression is tightly regulated and only transient on cells of the lymphoid lineage. In contrast, constitutive overexpression of CD70 has been documented in malignancies, where it appears to mediate immune escape through recruitment of CD27+ regulatory cells. Soluble CD27 (sCD27), the extracellular domain of CD27, can be released after lymphocyte activation and has been detected at increased levels in serum samples of lymphoid malignancies. To our knowledge, expression of CD70 has never been systematically characterized in non-small cell lung cancer (NSCLC). Methods: Fourty-nine primary NSCLC formalin-fixed paraffin embedded and 9 metastatic biopsies were stained by immunohistochemistry (IHC) for expression of CD70 (Clone 301731, 1/40) and CD27 (Clone 137B4, 1/40), using the Dako autostainer Link and Ventana ultra, respectively. Additionally, sCD27 levels were analyzed in 19 serum samples, taken before of biopsy, with the PeliKine Compact™ human sCD27 ELISA kit. Finally, we tested the efficacy of ARGX-110, a CD70-blocking monoclonal IgG1 SIMPLE Antibody™ endowed with enhanced ADCC properties (POTELLIGENT®), using the xCELLigence RTCA technology in CD70+ and CD70- cell lines. Results: In tumor cells, CD70 positivity (>10%) was detected in 16% of cases with a higher percentage in squamous NSCLC (27%) than in adenocarcinoma (9%). Additionally, one neuro-endocrine NSCLC was CD70+. We demonstrated identical CD70 patterns in primary and metastatic tissue in 8/10 biopsies. Furthermore, CD70 was found in the micro-environment of the tumor (23/49). IHC revealed high levels of CD27 expressing tumor infiltrating lymphocytes (TIL) in all NSCLC biopsies, with increasing FOXP3 expression and higher CD4/CD8 ratios in stromal tissue adjacent to CD70+ tumor cells. High sCD27 levels (>500U/ml) were found to be significantly associated with poor overall survival and even though sCD27 levels did not show potential as a blood-based biomarker for CD70 overexpression on tumor cells, dual positivity of CD70 and sCD27 marked even worse prognosis. We also showed that a low concentration of ARGX-110 (0.5μg/ml) induces efficient NK cell based tumor lysis in CD70+ cell lines. Conclusion: Expression of CD70 was demonstrated in tumor cells and TILs of NSCLC. Moreover, all biopsies revealed infiltration of CD27+ TIL in the micro-environment of the tumor and a trend towards increased FOXP3 expression and higher CD4/CD8 ratios in CD70+ biopsies. In addition, serum sCD27 levels have potential as a prognostic marker for NSCLC. Lastly, our in vitro results showed a maximum ADCC of ARGX-110 in CD70+ target cells stained by IHC. Hence, our data suggest that CD70 might be an interesting therapeutic target in NSCLC. Citation Format: Julie Jacobs, Patrick Pauwels, Christian Rolfo, Filip Lardon, Vanessa Deschoolmeester, Christophe Deben, Jolien Van den Bossche, Karen Zwaenepoel, Christophe Hermans, Karen Silence, Alain Thibault. Unlocking the potential of CD70 as a therapeutic target in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2015-3563